Tissue-engineered constructs, consisting of cells and 3-D scaffolds, have emerged as promising alternatives to biological and synthetic grafting materials for the repair of non-healing bone defects. However, host tissue-construct interactions, loss of osteoblastic phenotype under culture conditions, and limited supply of committed osteoprogenitor cells that will differentiate into osteoblasts restrict this approach. The objective of this application is to integrate cells genetically modified to express the osteoblast transcription factor Runx2/Cbfal into 3-D scaffolds to create hybrid constructs that promote matrix mineralization in vitro and in vivo. Our central hypothesis is that tissue-engineered constructs containing Runx2-expressing cells will significantly promote matrix mineralization in vitro and in vivo and enhance the healing of critical-size defects compared to constructs containing unmodified cells or empty scaffolds. The rationale for this work is that it will establish a genetic engineering strategy to overcome cell sourcing limitations associated with the application of tissue engineering to treat bone defects. Aim 1: Analyze the effects of Runx2 expression on gene/protein expression and matrix mineralization by cells cultured in 3-D polymeric scaffolds in vitro. We hypothesize that controlled expression of Runx2 enhances gene/protein expression and promotes matrix mineralization in 3-D constructs compared to unmodified cells. Aim 2: Examine the extent of bone-specific protein expression and mineralization in Runx2-engineered cells/scaffold constructs implanted into a subcutaneous site. We hypothesize that tissue-engineered constructs containing Runx2-modified cells exhibit enhanced in vivo ectopic bone formation compared to constructs containing unmodified cells. Aim 3: Evaluate the ability of tissue-engineered constructs containing Runx2-modified cells to repair critical-size bone defects. We will test the hypothesis that constructs containing Runx2-expressing cells promote in vivo bone formation and enhance repair of non-healing calvaria defects in syngeneic rats compared to cell-free scaffolds and constructs containing unmodified cells. This work is expected to yield the following outcomes: (1) establish the extent to which Runx2 expression enhances in vitro matrix mineralization and identify experimental parameters (scaffold pore size, dynamic culture conditions) that enhance mineralization in tissue-engineered constructs; (2) assess the ability of these constructs to form bone tissue in a non-osseous environment and provide information on the overall host response to these constructs; and (3) establish the potential of this genetic/tissue engineering approach to repair non-healing bone defects. Finally, these in vivo studies will also provide a solid foundation for validating the in vitro and subcutaneous models as surrogates for in vivo responses in an osseous environment. Collectively, these outcomes will validate this genetic engineering strategy for addressing cell sourcing issues in tissue engineering applications and establish the effectiveness of tissue engineering approaches to repair non-healing bone defects.